This work was supported by the Hebei Province Introducing Foreign Intelligence Project in 2023. We thank Lisa Kreiner, PhD, from Liwen Bianji, (Edanz) (www.liwenbianji.cn), for editing the English text of a draft of this manuscript.

The experimental procedure received approval from the Institutional Animal Care and Use Committee at Yantai University (Approval Number: YTDX20230410) and adhered to the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. Male Sprague-Dawley rats weighing between 300-350 g were utilized for this study. A comprehensive list of the animals, reagents, and equipment used is provided in the Table of Materials.
1. Animal preparation
<ol>
	<li>House the animals in an environment maintaining a 12 h light/dark cycle, controlled temperature, and humidity. Ensure they have unrestricted access to food and water.</li>
</ol>
2. Anesthesia
<ol>
	<li>Anesthetize the rats via intraperitoneal injection with a mixture containing ketamine (80 mg/kg) and xylazine (20 mg/kg). 
	NOTE: Tilt the head and body of rats downward. The injection site is in the animal&#8217;s lower right quadrant of the abdomen. Pull back on the plunger before injecting. The average injection volume is 0.4 mL.</li>
	<li>Confirm the induction of anesthesia by assessing the withdrawal reflex of the hind paw and the blinking reflex. If adequate depth of anesthesia is not achieved, supplement with isoflurane during the surgical procedure.</li>
</ol>
3. Ischemic modeling procedure
<ol>
	<li>Sterilize the surgical tools using an autoclave. Disinfect the operating tables and related equipment with 70% ethanol.</li>
	<li>Prepare the surgical site by shaving the fur around the anticipated incision site. Disinfect the skin at the surgical site three times with betadine and 70% ethanol.</li>
	<li>Make a midline incision on the ventral side, approximately 1.5 cm long, and open the superficial fascia.</li>
	<li>Perform sharp and blunt dissection to expose the external carotid artery (ECA), internal carotid artery (ICA), and common carotid artery (CCA) as per the previously described method7.</li>
	<li>Place three 4-0 silk sutures around the CCA and another silk suture through the ECA near the ICA bifurcation. Tie a releasable slip knot on the proximal CCA. Tighten the suture on the distal CCA using a hemostat to block blood flow. Loosely tie another slip knot around the CCA during suturing to ensure the vessel is not occluded. 
	NOTE: Ensure that the slip knot is not overly tight, only sufficient to block blood flow, as it will need to be released during vascular suturing.</li>
	<li>Make a 0.5 mm incision between the proximal silk suture and the middle suture on the CCA using scissors. 
	NOTE: Ensure that the incision allows the filament (silicon-coated tip) to enter the CCA.</li>
	<li>Introduce a 3 cm length of 4-0 filament (silicon-coated tip) into the CCA. Tighten the silk sutures around the CCA to secure the intraluminal filament and prevent bleeding; then, remove the suture from the distal CCA.</li>
	<li>Continue to insert the filament into the lumen of the MCA, controlling the sutures around the ECA to ensure the filament does not enter the ECA. The insertion length is typically 18 mm to 20 mm.</li>
	<li>Start a timer and record the time when occlusion begins. Suture the neck incision with 3-0 silk sutures and carefully place the animal in a recovery cage. Administer 0.5 mL of normal saline intraperitoneally.</li>
</ol>
4. Microsurgical suturing of the CCA
<ol>
	<li>Re-anesthetize the rat 1.5 h post-ischemia. Disinfect the incision site three times with 70% ethanol and reopen the incision.</li>
	<li>Gently withdraw the occluding filament until the white tip is clearly visible near the CCA incision. Place a micro clamp horizontally close to the secured intraluminal suture knot near the CCA bifurcation. 
	NOTE: Avoid clamping the white filament tip to prevent silicone detachment and subsequent thrombosis.</li>
	<li>Release the secured intraluminal suture knot and completely remove the filament.</li>
	<li>Place another micro clamp near the heart-end side of the proximal CCA knot. Subsequently, remove the suture knot around the proximal CCA. 
	NOTE: Proper placement of clamps is crucial for elevating fixed vessels in later steps to facilitate microvascular suturing. Place clamps horizontally on surrounding muscles to prevent slippage.</li>
	<li>Rinse the incision site with normal saline and dry the area with a sterile cotton ball to remove the blood and achieve a clear surgical field.</li>
	<li>Under a microscope (4x magnification), suture the CCA incision using 11-0 microsurgical sutures in an interrupted pattern, typically requiring two stitches depending on the incision size.</li>
	<li>After suturing, remove the micro clamps from both ends of the CCA to restore blood flow.</li>
	<li>Apply pressure with a sterile cotton ball to the CCA suture site for 30 s. Observe the CCA for 2 min to ensure no bleeding occurs. Suture the rat&#39;s neck incision and place the animal in a 35 &#176;C care box for anesthesia recovery before returning it to the cage. 
	NOTE: Administer intraperitoneally with meloxicam at a dose of 2 mg/kg for post-operative analgesia.</li>
</ol>
5. Staining with 2,3,5-triphenyltetrazolium chloride (TTC)
<ol>
	<li>Three days after the ischemia-reperfusion,&#160;euthanize the animals under deep anesthesia with 4% isoflurane and cervical dislocation.</li>
	<li>Remove the brains and cut them into 2 mm-thick coronal sections using a scalpel blade8.</li>
	<li>Incubate the brain sections with 2% TTC for 20 min at 37 &#176;C.</li>
	<li>Rinse the brain sections twice with 10% formalin, then fix them with 10% formalin for 30 min at room temperature.</li>
	<li>Capture digital photographs of consecutive slices and quantitatively measure the infarction areas using Image J software.</li>
</ol>
6. Neurological function test
NOTE: Neurological function was assessed using the Garcia test, following a previous report9.
<ol>
	<li>Assess spontaneous activity, symmetry in the movement of all four limbs, forepaw outstretching, climbing, body proprioception, and response to vibrissae touch. Assign scores based on the Garcia test criteria, with a maximum score of 18 points indicating normal neurological function.</li>
</ol>
7. Statistical analysis
<ol>
	<li>Utilize GraphPad Prism to conduct statistical analysis using Student&#39;s t-test. Express the data as the mean &#177; standard deviation. Consider differences between means statistically significant if P &#60; 0.05.</li>
</ol>

Middle Cerebral Artery Occlusion in Rats with Preserved Vascular Anatomy

<ol>
	<li>Tu, W. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=China+stroke+surveillance+report+2021.">China stroke surveillance report 2021.</a> Mil Med Res. 10 (1), 33 (2023).</li><li>Wu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+oxidative+stress+and+inflammation+to+prevent+ischemia-reperfusion+injury.">Targeting oxidative stress and inflammation to prevent ischemia-reperfusion injury.</a> Front in Mol Neurosci. 13, 28 (2020).</li><li>Ringelstein, E. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Type+and+extent+of+hemispheric+brain+infarctions+and+clinical+outcome+in+early+and+delayed+middle+cerebral+artery+recanalization.">Type and extent of hemispheric brain infarctions and clinical outcome in early and delayed middle cerebral artery recanalization.</a> Neurology. 42 (2), 289-298 (1992).</li><li>Koizumi, J., Yoshida, Y., Nakazawa, T., Ooneda, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+studies+of+ischemic+brain+edema:+A+new+experimental+model+of+cerebral+embolism+in+rats+in+which+recirculation+can+be+introduced+in+the+ischemic+area.">Experimental studies of ischemic brain edema: A new experimental model of cerebral embolism in rats in which recirculation can be introduced in the ischemic area.</a> Jap J Stroke. 8, 1-8 (1986).</li><li>Longa, E. Z., Weinstein, P. R., Carlson, S., Cummins, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversible+middle+cerebral+artery+occlusion+without+craniectomy+in+rats.">Reversible middle cerebral artery occlusion without craniectomy in rats.</a> Stroke. 20 (1), 84-91 (1989).</li><li>Trueman, R. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+critical+re-examination+of+the+intraluminal+filament+MCAO+model:+Impact+of+external+carotid+artery+transection.">A critical re-examination of the intraluminal filament MCAO model: Impact of external carotid artery transection.</a> Transl Stroke Res. 2 (4), 651-661 (2011).</li><li>Ansari, S., Azari, H., McConnell, D. J., Afzal, A., Mocco, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraluminal+middle+cerebral+artery+occlusion+(MCAO)+model+for+ischemic+stroke+with+laser+Doppler+flowmetry+guidance+in+mice.">Intraluminal middle cerebral artery occlusion (MCAO) model for ischemic stroke with laser Doppler flowmetry guidance in mice.</a> J Vis Exp. (51), e2879 (2011).</li><li>Yu, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Icaritin+inhibits+neuroinflammation+in+a+rat+cerebral+ischemia+model+by+regulating+microglial+polarization+through+the+GPER-ERK-NF-&#954;B+signaling+pathway.">Icaritin inhibits neuroinflammation in a rat cerebral ischemia model by regulating microglial polarization through the GPER-ERK-NF-&#954;B signaling pathway.</a> Mol Med. 28 (1), 142 (2022).</li><li>Garcia, J. H., Wagner, S., Liu, K. F., Hu, X. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurological+Deficit+and+extent+of+neuronal+necrosis+attributable+to+middle+cerebral+artery+occlusion+in+rats.">Neurological Deficit and extent of neuronal necrosis attributable to middle cerebral artery occlusion in rats.</a> Stroke. 26 (4), 627-634 (1995).</li><li>Bracko, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3-Nitropropionic+acid-induced+ischemia+tolerance+in+the+rat+brain+is+mediated+by+reduced+metabolic+activity+and+cerebral+blood+flow.">3-Nitropropionic acid-induced ischemia tolerance in the rat brain is mediated by reduced metabolic activity and cerebral blood flow.</a> J Cereb Blood Flow Metab. 34 (9), 1522-1530 (2014).</li><li>Goldmacher, G. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+transplanted+bone+marrow+stem+cells+and+their+effects+in+the+rat+MCAO+stroke+model.">Tracking transplanted bone marrow stem cells and their effects in the rat MCAO stroke model.</a> PLoS One. 8 (3), e60049 (2013).</li><li>Lee, H. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+therapeutic+potential+of+human+umbilical+cord+blood-derived+mesenchymal+stem+cells+in+Alzheimer's+disease.">The therapeutic potential of human umbilical cord blood-derived mesenchymal stem cells in Alzheimer's disease.</a> Neurosci Lett. 481 (1), 30-35 (2010).</li><li>Connolly, E. S., Winfree, C. J., Solomon, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Procedural+and+strain-related+variables+significantly+affect+outcome+in+a+murine+model+of+focal+cerebral+ischemia.">Procedural and strain-related variables significantly affect outcome in a murine model of focal cerebral ischemia.</a> Neurosurgery. 38 (3), 523-531 (1996).</li><li>Barone, F. C., Knudsen, D. J., Nelson, A. H., Feuerstein, G. Z., Willette, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+strain+differences+in+susceptibility+to+cerebral+ischemia+are+related+to+cerebral+vascular+anatomy.">Mouse strain differences in susceptibility to cerebral ischemia are related to cerebral vascular anatomy.</a> J Cereb Blood Flow Metab. 13 (4), 683-692 (1993).</li><li>Dittmar, M., Spruss, T., Schuierer, G., Horn, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=External+carotid+artery+territory+ischemia+impairs+outcome+in+the+endovascular+filament+model+of+middle+cerebral+artery+occlusion+in+rats.">External carotid artery territory ischemia impairs outcome in the endovascular filament model of middle cerebral artery occlusion in rats.</a> Stroke. 34 (9), 2252-2257 (2003).</li><li>Virtanen, T., Jolkkonen, J., Sivenius, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=External+carotid+artery+territory+ischemia+impairs+outcome+in+the+endovascular+filament+model+of+middle+cerebral+artery+occlusion+in+rats.">External carotid artery territory ischemia impairs outcome in the endovascular filament model of middle cerebral artery occlusion in rats.</a> Stroke. 34 (9), 2252-2257 (2004).</li></ol>

The authors do not have any potential conflicting interests to disclose.

MCAo is a widely used method to create animal models of stroke, effectively replicating the stroke process observed in patients by restoring blood flow after ischemia10,11,12. Our laboratory has developed an ischemic stroke model that enables reperfusion from the CCA without causing damage to the ECA. Following the induction of ischemia, microsurgical repair of the CCA is performed. Although direct measurement of CCA blood flow was not conducted, experimenters observed vascular pulsation in the CCA before proceeding with reperfusion. This technique preserves the anatomical integrity of the neck vessels while closely resembling the stroke process in patients. Furthermore, evaluations of neurological function and the area of brain ischemia have confirmed the validity of the stroke induced by this surgical approach.
The previously described ischemic stroke models can be affected by various factors. In the method by Koizumi et al.4, blood reperfusion heavily relies on the circle of Willis due to the ligation of the CCA. However, variations in cerebral vasculature can influence the final cerebral infarction volume, potentially increasing the mortality rate of the stroke model due to low blood perfusion13,14. Additionally, ischemia of the ECA can impair the function of facial muscles and nerves, impacting the assessment of various neurological functions15,16.
The current method addresses these challenges through three key steps: (1) Keeping the CCA incision as small as possible, only large enough for filament insertion, reducing time and damage associated with vascular suturing. (2) Placing microvascular clamps as close to the CCA bifurcation as possible when clamping the distal CCA to prevent clamping the white filament tip, which could lead to silicone detachment and thrombosis. (3) Observing the CCA for resumed pulsation and lack of substantial bleeding before suturing the midline neck incision after vascular repair.
The current method does have several limitations. Firstly, there is a possibility of forming emboli at the suture site, which could lead to further brain ischemia if they detach. This risk should be carefully considered and managed in future experiments. Secondly, the study did not monitor the body weight of the rats, which makes it challenging to determine if the method affected their food intake. Food intake is a direct indicator of the method&#39;s advantages, and its assessment could provide valuable insights. Lastly, the current method requires technical proficiency in microsurgical operations, which may limit its widespread use without proper training and expertise.
In summary, while the MCAo modeling method offers high physiological relevance and structural integrity, it does come with limitations that need to be addressed for more robust experimentation. Preserving the anatomical integrity of neck vessels and closely simulating stroke processes in patients are significant strengths of the method. This approach will undoubtedly contribute significantly to ischemic stroke research, improving the reliability and accuracy of experimental models. Ultimately, this may lead to fewer experimental groups needed to test therapeutic effects, resulting in more reliable experimental outcomes.

Stroke, the leading cause of death and long-term neurological dysfunction in adults worldwide, encompass various types, with ischemic stroke accounting for approximately 81.9% of cases, while cerebral hemorrhage and subarachnoid hemorrhage account for 14.9% and 3.1%, respectively1. Ischemic stroke often results from atherosclerosis or occlusion of the middle cerebral artery (MCA), leading to local cerebral blood flow reduction and subsequent brain damage. This reduction in nutrient supply to ischemic cells triggers energy depletion, causing membrane integrity loss, cell swelling, and eventual lysis. Additionally, factors like increased intracellular calcium, excitatory amino acid release, heightened free radical production, and inflammatory cell activation contribute to brain tissue injury post-stroke2.
The treatment goals for ischemic stroke are multifaceted: reduce ongoing neurological damage and lower mortality and long-term disability; prevent complications arising from immobility and neurological dysfunction; and minimize the risk of stroke recurrence. Given the complexity of ischemic stroke pathophysiology, a robust stroke model is essential for the research and development of treatments targeting ischemic stroke.
Currently, middle cerebral artery occlusion (MCAo) is a common animal model of ischemic stroke3. MCAo is typically performed by inserting a silicon-tipped or flame-blunted monofilament through the cervical vessels until it reaches the origin of the middle cerebral artery (MCA). In 1986, Koizumi and colleagues developed a technique involving the insertion of a silicon-tipped monofilament through an incision in the common carotid artery (CCA), allowing it to reach the entrance of the MCA and block its blood flow4. In 1989, Longa's group reported another MCAo method that involved introducing a monofilament through an incision in the external carotid artery (ECA). The monofilament then traverses the bifurcation of the internal carotid artery (ICA) and ECA before reaching the starting point of the MCA5.
The limitations of the two existing methods for inducing MCAo are evident. Koizumi's method necessitates the permanent blockage of the ipsilateral CCA, leading to reduced MCA perfusion since reperfusion solely relies on the circle of Willis4. Conversely, Longa's method involves cutting the ECA, resulting in damage to the ECA system and subsequent impairment of facial muscle function5. This damage can impact food and water intake, as well as cause sensory dysfunction of the face, affecting rehabilitation and the assessment of neurological function post-ischemic stroke6.
The current protocol seeks to address these limitations by developing a new method to induce an ischemic stroke model without damaging the anatomical structure of cerebral vessels. This novel approach utilizes microsurgical techniques to repair the CCA, thus avoiding the drawbacks associated with previous methods.

<table><tbody><tr><td>10% formalin</td><td>Beijing Labgic Technology Co.,Ltd.</td><td>BL913A</td><td></td></tr><tr><td>11-0 microsurgical sutures</td><td>Ningbo Medical Suture Needle Co., Ltd.</td><td>3/8 1&amp;times;5</td><td></td></tr><tr><td>2,3,5-triphenyltetrazolium chloride (TTC)</td><td>SIGMA</td><td>T8877</td><td></td></tr><tr><td>3-0 silk suture</td><td>Ningbo Medical Suture Needle Co., Ltd.</td><td>3-0</td><td></td></tr><tr><td>4% isoflurane&amp;nbsp;&amp;nbsp;</td><td>Tianjin Ringpu Bio-Technology Co., Ltd.</td><td>20221102</td><td></td></tr><tr><td>6-0 silk suture</td><td>Ningbo Medical Suture Needle Co., Ltd.</td><td>6-0</td><td></td></tr><tr><td>70% ethanol</td><td>Shandong Lierkang Medical Technology Co., Ltd.</td><td>20230408</td><td></td></tr><tr><td>Filament</td><td>Xinong Technologies Co Ltd.</td><td>2838-A5</td><td></td></tr><tr><td>Ketamine&amp;nbsp;&amp;nbsp;</td><td>Jiangsu Hengrui Pharmaceutical Co., Ltd.</td><td>230613BL</td><td></td></tr><tr><td>Male Sprague Dawley rats</td><td>Jinan Pengyue Experimental Animal Breeding Co., Ltd.</td><td>SCXK (Lu) 20190003</td><td></td></tr><tr><td>Microclips</td><td>Shanghai Jinzhong Medical Device Factory</td><td>W40150</td><td></td></tr><tr><td>Microscope</td><td>ZEISS</td><td>S100 OPMI pico</td><td></td></tr><tr><td>Nursing box</td><td>Beijing Fuyi Electrical Appliance Co., Ltd.</td><td>FYL-YS-100L</td><td></td></tr><tr><td>Xylazine</td><td>SIGMA</td><td>PHR3263</td><td></td></tr></tbody></table>

a rat model of middle cerebral artery occlusion/reperfusion without damaging the anatomical structure of cerebral vessels

Ischemic stroke stands as the primary cause of long-term disability and mortality among adults worldwide. Animal models of ischemic stroke have significantly contributed to our understanding of its pathological mechanisms and the development of potential treatments. Presently, there are two common methods involving filament (endovascular suture) techniques to induce animal models of cerebral ischemia. However, these methods have inherent limitations, such as reduced blood perfusion to the brain, damage to the external carotid artery system, impaired food and/or water intake, and sensory dysfunction of the face. This article introduces a new method for inducing a rat ischemic stroke model without compromising the cerebral vascular anatomy. In this study, the common carotid artery (CCA) of Sprague-Dawley rats was exposed, and an incision was made. A filament was then inserted through the incision into the internal carotid artery to occlude the middle cerebral artery. After 1.5 h of induced ischemia, the occluding filament was fully removed from both the internal carotid artery and the CCA. The incision in the CCA was subsequently sutured using 11-0 microsurgical sutures under a microscope (magnification 4x). Through the utilization of microsurgical techniques to repair the CCA, this study successfully developed a unique method to induce an ischemic stroke model in rats while preserving the anatomical integrity of cerebral blood vessels.

The process of microsuture 
The filament was withdrawn near the incision of the CCA until the white filament tip was visible (Figure 1A). Micro clamps were then placed on the CCA (Figure 1B). Under a microscope at 4x magnification, the incision of the CCA was sutured using microsurgical sutures in an interrupted pattern (Figure 1C). Subsequently, the microclamps were removed from the CCA, restoring the anatomical structure and blood flow (Figure 1D).
TTC staining and mortality rate 
A total of 12 adult male rats were randomly divided into two groups: sham and model (n = 6 in each group). Unfortunately, one animal in the sham group died from an anesthetic overdose, and one animal in the model group died within 24 h. The infarcts resulting from cerebral ischemia were observed in both the striatum and the cortex (Figure 2A). At 3 days post-ischemic stroke, the total infarct percentage was measured to be 29.3% &#177; 1.6% of the hemisphere (Figure 2B), with a mortality rate of 16.7% after surgery.
Evaluation of neurological function 
At 1 and 3 days post-ischemic stroke, the scores of the model groups significantly decreased compared to those of the sham groups (17.8 &#177; 0.4 vs. 8.2 &#177; 1.5, Figure 3A; 17.6 &#177; 0.9 vs. 9.6 &#177; 1.3, Figure 3B, respectively).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66635/66635fig01.jpg" /> 
Figure 1: The process of microsuture. (A) The filament is withdrawn from the CCA. Blue arrows indicate the microvascular clamps. (B) Black arrow indicates the incision after the filament is completely removed. Blue arrows indicate the microvascular clamps. (C) Under a microscope (magnified 4x), the CCA incision was sutured using 11-0 microsurgical sutures in an interrupted pattern. Blue arrows indicate the microvascular clamps. Black arrow indicates the sutured incision. (D) The micro clamps are removed and the blood flow of CCA is restored. Black arrow indicates the sutured site of CCA. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66635/66635fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66635/66635fig02.jpg" /> 
Figure 2: TTC staining results of the ischemia model. (A) Representative images of 2,3,5-triphenyltetrazolium chloride (TTC)-stained brain slices after cerebral ischemia. In living tissue, TTC is enzymatically reduced by dehydrogenases to 1,3,5-triphenylformazan, which has a red color. The tissues in necrotic areas remain white because of the absence of such enzymatic activity. (B) Percentage of ischemic area in rats at 3 days after cerebral ischemia. Compared with the Sham group, **P &#60; 0.01. Data are expressed as the mean &#177; standard deviation. There were five rats in each group. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66635/66635fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66635/66635fig03.jpg" /> 
Figure 3: Evaluation of neurological function. (A) Garcia scores 1 day after cerebral ischemia. (B) Garcia scores 3 days after cerebral ischemia. Compared with the Sham group, **P &#60; 0.01. Data are expressed as mean &#177; standard deviation. There were five rats in each group. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66635/66635fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Read this Scientific Article Protocol about A Rat Model of Middle Cerebral Artery Occlusion/Reperfusion without Damaging the Anatomical Structure of Cerebral Vessels at JoVE.com

A Rat Model of Middle Cerebral Artery Occlusion/Reperfusion Without Damaging the Anatomical Structure of Cerebral Vessels

The current protocol describes a rat model of middle cerebral artery occlusion/reperfusion that preserves the anatomical structure of cerebral vessels without causing damage.

Ischemic stroke stands as the primary cause of long-term disability and mortality among adults worldwide. Animal models of ischemic stroke have ...

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378 Aufrufe.  The Third Hospital of Hebei Medical University. The current protocol describes a rat model of middle cerebral artery occlusion/reperfusion that preserves the anatomical structure of cerebral vessels without causing damage.

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Modeling Stroke in Mice - Middle Cerebral Artery Occlusion with the Filament Model

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are very limited. To meet the need for novel therapeutic approaches, experimental stroke research frequently employs rodent models of focal cerebral ischaemia. Most researchers use permanent or transient occlusion of the middle cerebral artery (MCA) in mice or rats.
Proximal occlusion of the middle cerebral artery (MCA) via the intraluminal suture technique (so called filament or suture model) is probably the most frequently used model in experimental stroke research. The intraluminal MCAO model offers the advantage of inducing reproducible transient or permanent ischaemia of the MCA territory in a relatively non-invasive manner. Intraluminal approaches interrupt the blood flow of the entire territory of this artery. Filament occlusion thus arrests flow proximal to the lenticulo-striate arteries, which supply the basal ganglia. Filament occlusion of the MCA results in reproducible lesions in the cortex and striatum and can be either permanent or transient. In contrast, models inducing distal (to the branching of the lenticulo-striate arteries) MCA occlusion typically spare the striatum and primarily involve the neocortex. In addition these models do require craniectomy. In the model demonstrated in this article, a silicon coated filament is introduced into the common carotid artery and advanced along the internal carotid artery into the Circle of Willis, where it blocks the origin of the middle cerebral artery. In patients, occlusions of the middle cerebral artery are among the most common causes of ischaemic stroke. Since varying ischemic intervals can be chosen freely in this model depending on the time point of reperfusion, ischaemic lesions with varying degrees of severity can be produced. Reperfusion by removal of the occluding filament at least partially models the restoration of blood flow after spontaneous or therapeutic (tPA) lysis of a thromboembolic clot in humans.
In this video we will present the basic technique as well as the major pitfalls and confounders which may limit the predictive value of this model.

Filamentous occlusion of the Middle cerebral artery is a common model for studying ischemic stroke in mice.

Modeling Stroke in Mice - Middle Zerebralarterie mit dem Filament-Modell

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are ...

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Medizin

Mouse Model of Middle Cerebral Artery Occlusion

Stroke is the most common fatal neurological disease in the United States 1. The majority of strokes (88%) result from blockage of blood vessels in the brain (ischemic stroke) 2. Since most ischemic strokes (~80%) occur in the territory of middle cerebral artery (MCA) 3, many animal stroke models that have been developed have focused on this artery. The intraluminal monofilament model of middle cerebral artery occlusion (MCAO) involves the insertion of a surgical filament into the external carotid artery and threading it forward into the internal carotid artery (ICA) until the tip occludes the origin of the MCA, resulting in a cessation of blood flow and subsequent brain infarction in the MCA territory 4. The technique can be used to model permanent or transient occlusion 5. If the suture is removed after a certain interval (30 min, 1 h, or 2 h), reperfusion is achieved (transient MCAO); if the filament is left in place (24 h) the procedure is suitable as a model of permanent MCAO. This technique does not require craniectomy, a neurosurgical procedure to remove a portion of skull, which may affect intracranial pressure and temperature 6. It has become the most frequently used method to mimic permanent and transient focal cerebral ischemia in rats and mice 7,8. To evaluate the extent of cerebral infarction, we stain brain slices with 2,3,5-triphenyltetrazolium chloride (TTC) to identify ischemic brain tissue 9. In this video, we demonstrate the MCAO method and the determination of infarct size by TTC staining.

We demonstrate in the video a method for producing a middle cerebral artery occlusion in adult mice using an intraluminal monofilament. We also show how to evaluate the extent of cerebral infarction by 2,3,5-triphenyltetrazolium chloride (TTC) staining.

Mouse Model of Middle Zerebralarterie

Stroke is the most common fatal neurological disease in the United States 1. The majority of strokes (88%) result from blockage of blood vessels in the ...

Permanent Cerebral Vessel Occlusion via Double Ligature and Transection

Stroke is a leading cause of death, disability, and socioeconomic loss worldwide. The majority of all strokes result from an interruption in blood flow (ischemia) 1. Middle cerebral artery (MCA) delivers a great majority of blood to the lateral surface of the cortex 2, is the most common site of human stroke 3, and ischemia within its territory can result in extensive dysfunction or death 1,4,5. Survivors of ischemic stroke often suffer loss or disruption of motor capabilities, sensory deficits, and infarct. In an effort to capture these key characteristics of stroke, and thereby develop effective treatment, a great deal of emphasis is placed upon animal models of ischemia in MCA.
Here we present a method of permanently occluding a cortical surface blood vessel. We will present this method using an example of a relevant vessel occlusion that models the most common type, location, and outcome of human stroke, permanent middle cerebral artery occlusion (pMCAO). In this model, we surgically expose MCA in the adult rat and subsequently occlude via double ligature and transection of the vessel. This pMCAO blocks the proximal cortical branch of MCA, causing ischemia in all of MCA cortical territory, a large portion of the cortex. This method of occlusion can also be used to occlude more distal portions of cortical vessels in order to achieve more focal ischemia targeting a smaller region of cortex. The primary disadvantages of pMCAO are that the surgical procedure is somewhat invasive as a small craniotomy is required to access MCA, though this results in minimal tissue damage. The primary advantages of this model, however, are: the site of occlusion is well defined, the degree of blood flow reduction is consistent, functional and neurological impairment occurs rapidly, infarct size is consistent, and the high rate of survival allows for long-term chronic assessment.

Permanent Cerebral Vessel Occlusion via Double Ligature and Transection

We describe a highly reproducible method for the permanent occlusion of a rodent major cerebral blood vessel. This technique can be accomplished with very little peripheral damage, minimal blood loss, a high rate of long-term survival, and consistent infarct volume commensurate with the human clinical population.

Permanent Cerebral Gefäßverschluss<em&gt; Über</em&gt; Double Blattschraube und Durchtrennung

Stroke is a leading cause of death, disability, and socioeconomic loss worldwide. The majority of all strokes result from an interruption in blood flow ...

Modeling Stroke in Mice: Permanent Coagulation of the Distal Middle Cerebral Artery

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical options are available for a small proportion of stroke patients in the acute phase. Current research is intensively searching for novel therapeutic strategies and is increasingly focusing on the sub-acute and chronic phase after stroke because more patients might be eligible for therapeutic interventions in a prolonged time window. These delayed mechanisms include important pathophysiological pathways such as post-stroke inflammation, angiogenesis, neuronal plasticity and regeneration. In order to analyze these mechanisms and to subsequently evaluate novel drug targets, experimental stroke models with clinical relevance, low mortality and high reproducibility are sought after. Moreover, mice are the smallest mammals in which a focal stroke lesion can be induced and for which a broad spectrum of transgenic models are available. Therefore, we describe here the mouse model of transcranial, permanent coagulation of the middle cerebral artery via electrocoagulation distal of the lenticulostriatal arteries, the so-called &#8220;coagulation model&#8221;. The resulting infarct in this model is located mainly in the cortex; the relative infarct volume in relation to brain size corresponds to the majority of human strokes. Moreover, the model fulfills the above-mentioned criteria of reproducibility and low mortality. In this video we demonstrate the surgical methods of stroke induction in the &#8220;coagulation model&#8221; and report histological and functional analysis tools.

Various murine models of middle cerebral artery occlusion (MCAO) are widely used in experimental brain research. Here, we demonstrate the model of transcranial permanent distal MCAO which produces consistent cortical infarction of a size corresponding to damage imposed by the majority of human ischemic strokes.

Modellierung Schlaganfall bei Mäusen: Permanent Koagulation der distalen A. cerebri media

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical ...

Surgical Approach for Middle Cerebral Artery Occlusion and Reperfusion Induced Stroke in Mice

Stroke is a leading cause of death worldwide and continues to be one of the major causes of long-term adult disabilities. About 87% of strokes are ischemic in origin and occur in the territory of the middle cerebral artery (MCA). Currently the only Food and Drug Administration (FDA) approved drug for the treatment of this devastating disease is tissue plasminogen activator (tPA). However, tPA has a small therapeutic window for administration (3 - 6 hr), and is only effective in 4% of the patients who actually receive it. Current research focuses on understanding the pathophysiology of stroke in order to find potential therapeutic targets. Thus, reliable models are crucial, and the MCA occlusion (MCAo) model (also termed the intraluminal filament or suture model) is deemed to be the most clinically relevant surgical model of ischemic stroke, and is fairly non-invasive and easily reproducible. Typically the MCAo model is used with rodents, especially with mice due to all the genetic variations available for this species. Here we describe (and present in the video) how to successfully perform the MCAo model (with reperfusion) in mice to generate reliable and reproducible data.

In order to understand the pathophysiology of stroke, it is important to use reliable models. This paper will describe one of the most frequently used stroke models in mice, termed the middle cerebral artery occlusion (MCAo) model (also termed the intraluminal filament or suture model) with reperfusion.

Chirurgische Ansatz für Arteria cerebri media Occlusion und Reperfusion fokaler zerebraler Ischämie bei Mäusen

Stroke is a leading cause of death worldwide and continues to be one of the major causes of long-term adult disabilities. About 87% of strokes are ...

Transient Middle Cerebral Artery Occlusion Model of Neonatal Stroke in P10 Rats

A number of animal models have been used to study hypoxic-ischemic injury, traumatic injury, global hypoxia, or permanent ischemia in both the immature and mature brain. Stroke occurs commonly in the perinatal period in humans, and transient ischemia-reperfusion is the most common form of stroke in neonates. The reperfusion phase is a critical component of injury progression, which occurs over a period of days to weeks, and of the endogenous response to injury. This postnatal day 10 (p10) rat model of transient middle cerebral artery occlusion (tMCAO) creates a unilateral, non-hemorrhagic focal ischemia-reperfusion injury that can be utilized to study the mechanisms of focal injury and repair in the full-term-equivalent brain. The injury pattern that is produced by tMCAO is consistent and highly reproducible and can be confirmed with MRI or histological analyses. The severity of injury can be manipulated through changes in occlusion time and other methods that will be discussed.

Neonatal stroke is a significant cause of early brain injury requiring a translational model with consistent focal injury patterns and high reproducibility in order to enable study. This study describes the detailed surgical procedure for creating a non-hemorrhagic, unilateral focal ischemia-reperfusion injury in full-term-equivalent rodents.

Transient Middle Cerebral Artery Occlusion Modell von Neugeborenen Stroke in P10 Ratten

A number of animal models have been used to study hypoxic-ischemic injury, traumatic injury, global hypoxia, or permanent ischemia in both the immature ...

Middle Cerebral Artery Occlusion Allowing Reperfusion via Common Carotid Artery Repair in Mice

The ischemic stroke is a major cause of adult long-term disability and death worldwide. The current treatments available are limited, with only tissue plasminogen activator (tPA) as an approved drug treatment to target ischemic strokes. Current research in the field of ischemic stroke focuses on better understanding the pathophysiology of stroke, to develop and investigate novel pharmaceutical targets. Reliable experimental stroke models are crucial for the progression of potential treatments. The middle cerebral artery occlusion (MCAO) model is clinically relevant and the most frequently used surgical model of ischemic stroke in rodents. However, the outcomes of this model, such as lesion volume, are associated with high levels of variability, particularly in mice. The alternative MCAO model described here allows the reperfusion of the common carotid artery (CCA) and the increased perfusion of the middle cerebral artery (MCA) territory, using a tissue pad with fibrinogen-based sealant to repair the vessel, and the improved welfare of the mice by avoiding external carotid artery (ECA) ligation. This reduces the reliance on the Circle of Willis, which is known to be highly anatomically variable in mice. Representative data show that using this alternative surgical approach decreases the variability in lesion volumes between the traditional MCAO approach and the alternative approach described here.

Intraluminal filament occlusion of the middle cerebral artery is the most frequently used in vivo model of experimental stroke in rodents. An alternative surgical approach to allow common carotid artery repair is performed here, which allows the reperfusion of the common carotid artery and a full reperfusion to the middle cerebral artery territory.

Mittlere zerebrale Arterie Okklusion so dass Reperfusion über gemeinsame Halsschlagader Reparatur bei Mäusen

The ischemic stroke is a major cause of adult long-term disability and death worldwide. The current treatments available are limited, with only tissue ...

Two-vessel Occlusion Mouse Model of Cerebral Ischemia-reperfusion

In this study, a middle cerebral artery (MCA) occlusion mouse model is employed to study cerebral ischemia-reperfusion. A reproducible and reliable mouse model is useful for investigating the pathophysiology of cerebral ischemia-reperfusion and determining potential therapeutic strategies for patients with stroke. Variations in the anatomy of the circle of Willis of C57BL/6 mice affects their infarct volume after cerebral-ischemia-induced injury. Studies have indicated that distal MCA occlusion (MCAO) can overcome this problem and result in a stable infarct size. In this study, we establish a two-vessel occlusion mouse model of cerebral ischemia-reperfusion through the interruption of the blood flow to the right MCA. We distally ligate the right MCA and right common carotid artery (CCA) and restore blood flow after a certain period of ischemia. This ischemia-reperfusion injury induces an infarct of stable size and a behavioral deficit. Peripheral immune cells infiltrate the ischemic brain within the 24 h infiltration period. Additionally, the neuronal loss in the cortical area is less for a longer reperfusion duration. Therefore, this two-vessel occlusion model is suitable for investigating the immune response and neuronal recovery during the reperfusion period after cerebral ischemia.

A mouse model of cerebral ischemia-reperfusion is established to investigate the pathophysiology of stroke. We distally ligate the right middle cerebral artery and right common carotid artery and restore blood flow after 10 or 40 min of ischemia.

Zwei-Schiff Okklusion Mausmodell der zerebralen Ischämie-reperfusion

In this study, a middle cerebral artery (MCA) occlusion mouse model is employed to study cerebral ischemia-reperfusion. A reproducible and reliable mouse ...

Ein Modell der transitorischen zerebralen Ischämie durch Verschluss der Arteria cerebralis media

Transient Middle Cerebral Artery Occlusion Model of Stroke

Stroke stands as a major cause of death or chronic disability globally. Nevertheless, existing optimal treatments are limited to reperfusion therapies during the acute phase of ischemic stroke. To gain insights into stroke physiopathology and develop innovative therapeutic approaches, in vivo rodent models of stroke play a fundamental role. The availability of genetically modified animals has particularly propelled the use of mice as experimental stroke models.
In stroke patients, occlusion of the middle cerebral artery (MCA) is a common occurrence. Consequently, the most prevalent experimental model involves intraluminal occlusion of the MCA, a minimally invasive technique that doesn't require craniectomy. This procedure involves inserting a monofilament through the external carotid artery (ECA) and advancing it through the internal carotid artery (ICA) until it reaches the branching point of the MCA. After a 45 min arterial occlusion, the monofilament is removed to allow reperfusion. Throughout the process, cerebral blood flow is monitored to confirm the reduction during occlusion and subsequent recovery upon reperfusion. Neurological and tissue outcomes are evaluated using behavioral tests and magnetic resonance imaging (MRI) studies.

Autor im Rampenlicht: Bewertung von ischämischen Schlaganfallschäden durch ein Modell zur Okklusion der mittleren Hirnarterie 

This protocol describes the model of transient focal cerebral ischemia in mice through intraluminal occlusion of the middle cerebral artery. Additionally, examples of outcome assessment are shown using magnetic resonance imaging and behavioral tests.

Transienter mittlerer Hirnarterienverschluss Modell des Schlaganfalls

Stroke stands as a major cause of death or chronic disability globally. Nevertheless, existing optimal treatments are limited to reperfusion therapies ...

A Rat Model of Middle Cerebral Artery Occlusion/Reperfusion Without Damaging the Anatomical Structure of Cerebral Vessels

Zusammenfassung

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